肺炎链球菌毒力蛋白基因工程疫苗的实验研究
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摘要
背景与目的:
肺炎链球菌是社区获得性感染最常见的致病菌,全球每年有超过100万儿童死于肺炎链球菌感染性疾病。目前肺炎链球菌对抗生素多重耐药情况日趋严重,使肺炎链球菌感染的治疗面临重大挑战,WHO提出应用疫苗预防肺炎链球菌感染是减少耐药菌传播、缓解抗生素耐药压力,保护高危人群的唯一方法。
国内外越来越多的临床研究发现目前应用的肺炎链球菌23价荚膜多糖疫苗和7价蛋白-多糖结合疫苗存在血清型依赖,成本昂贵,不降低肺炎链球菌鼻咽部携带等不足。研发新一代非血清型依赖的安全、便携、廉价的新型肺炎链球菌疫苗具有巨大的经济效益和深远的社会价值。
肺炎链球菌作为条件致病菌,多种毒力因子独立或协同的参与了其粘附、定植、迁移、侵袭性致病的全过程。基于肺炎链球菌相关毒力蛋白的DNA疫苗具有非血清型依赖、覆盖率广、造价低廉等优势,成为第三代肺炎链球菌疫苗的研究热点。靶抗原的选择,优势载体系统的应用和理想的接种途径是新型肺炎链球菌DNA疫苗研究的关键环节。本课题组前期研究已经证实了肺炎链球菌表面蛋白A(pneumococcal surface protein A,PspA)及肺炎链球菌黏附素A(pneumococcal surface adhesion A ,PsaA)的联合是最优势的候选抗原组合形式,但是传统的DNA疫苗免疫途径并不具备激发鼻咽部特异性sIgA抗体保护的能力,而且免疫原性较弱。鉴于鼻咽部定植是肺炎链球菌感染的首要步骤,研究如何优化DNA疫苗的载体系统,选择理想的免疫途径进而增强肺炎链球菌DNA疫苗的免疫效能,尤其是增加其鼻咽部粘膜免疫保护效能具有重要意义。
因此,本课题围绕“肺炎链球菌基因工程疫苗的实验研究”,进行了以下三部分研究工作:(1)第一部分:基于优势靶抗原组合(pspA+psaA基因),构建肺炎链球菌多价DNA疫苗。复制肺炎链球菌鼻咽部携带及腹腔攻击动物模型,裸质粒肌肉注射方式免疫接种,评价其免疫原性及保护效能;(2)第二部分:基于载体-宿主致死平衡原理,设计改造现有减毒沙门菌为宿主菌的沙门菌-原核质粒平衡致死系统,将其携带的原核质粒改造为非抗性基因筛选的真核质粒,构建新型减毒沙门菌为载体的多价肺炎链球菌DNA基因工程疫苗,复制肺炎链球菌鼻咽部携带及腹腔攻击动物模型,经口服途径免疫接种,评价其稳定性、免疫原性及免疫保护效能;(3)第三部分:进一步利用基因组学及分子遗传学技术针对优势靶抗原进行分子进化及比较基因组学的初步研究。旨在为肺炎链球菌新一代基因工程疫苗的研发提供理论基础和实验室依据。方法:
1.分子克隆技术构建PsaA和PspA’(PspA的N端抗原表位所在区域基因片段)的重组真核表达质粒PsaA-pcDNA3.1和PspA’-pcDNA3.1。两种重组质粒DNA疫苗PsaA-pcDNA3.1和PspA’-pcDNA3.1裸质粒单独或联合肌肉注射免疫小鼠,ELISA检测免疫小鼠脾细胞分泌细胞因子及血清中特异性抗体IgG水平;复制小鼠肺炎链球菌D39株鼻咽部携带和腹腔侵袭性感染模型,观察免疫小鼠鼻咽部肺炎链球菌携带改变和腹腔攻击小鼠21天生存情况。
2.新型减毒沙门菌为载体的多价肺炎链球菌DNA疫苗构建及其稳定性、免疫效能检测。
(1)肺炎链球菌DNA疫苗的新型减毒沙门菌载体系统构建:基于pcDNA3.1(+),应用BspHI、XmnⅠ双酶切获得片段SV40ori-Neor-SV40Pa-pUCori和Pcmv-MCS-BGHpA-f1ori,再用SalⅠ酶切SV40ori-Neor-SV40pA-pUCori获得片段pUCori;将酶切获得的pUCori、Pcmv-MCS-BGHpA-f1ori两个片段及变形链球菌asd基因PCR扩增产物进行连接,构建新互补质粒pcDNA3.1000,并最终转化入宿主菌沙门菌χ4550中(pcDNA3.1000x)。
(2)分子克隆技术构建新型减毒沙门菌为载体的肺炎链球菌DNA疫苗pcDNA3.1001x(编码pspA’基因)和pcDNA3.1002x(编码psaA基因),并检测重组质粒在哺乳动物细胞BHK-21中的表达(RT-PCR及Western-blot)及质粒稳定性。新型减毒沙门菌为载体的肺炎链球菌DNA疫苗pcDNA3.1001x和pcDNA3.1002x单独或联合口服免疫小鼠,ELISA检测免疫小鼠脾细胞分泌的细胞因子、血清中特异性抗体IgG及鼻咽部灌洗液中sIgG水平。复制小鼠肺炎链球菌D39株鼻咽部携带模型和腹腔侵袭性感染模型,观察免疫小鼠鼻咽部肺炎链球菌D39菌落计数改变和腹腔攻击小鼠21天生存情况,以评价新型减毒沙门菌为载体的多价肺炎链球菌DNA疫苗的免疫原性及保护作用优势。
3.采用分子遗传学技术进一步分析肺炎链球菌基因工程疫苗优势抗原蛋白的分子进化特点及抗原多样性。
(1)采集上呼吸道共栖环境中的临床收集轻链球菌家族菌株(包含肺炎链球菌、轻链球菌、口腔链球菌、血链球菌、婴儿链球菌),PCR技术探查psaA基因分布情况。基于扩增的psaA基因进行氨基酸序列比对及系统发生树构建,分析psaA的序列特点及在轻链球菌家族中可能的遗传机制。
(2)采集本地区临床致病肺炎链球菌感染菌株,应用PspA蛋白家族分型特异性引物PCR及生物信息学技术鉴定本地区临床自然感染肺炎链球菌株PspA家族分布特点;进一步应用分子遗传学技术构建pspA基因多家族的系统发生树,分析肺炎链球菌PspA作为新一代肺炎链球菌基因工程疫苗优势候选抗原应包含的优势家族成分。
结果:
1.肺炎链球菌多价DNA疫苗的构建:成功扩增pspA基因包含抗原表位的N端片段及psaA基因目标片段,分别克隆入真核载体pcDNA3.1(+),构建了PspA’-pcDNA3.1和PsaA-pcDNA3.1两种肺炎链球菌毒力蛋白重组真核质粒,并从转录水平和翻译水平检测了这两种重组质粒编码的psaA及pspA’基因在哺乳动物细胞的成功表达。
2.肺炎链球菌多价DNA疫苗的免疫原性及保护效能
(1)免疫原性评价
①细胞免疫评价:单独及联合免疫组小鼠脾细胞分泌的抗原特异性细胞因子IFN-γ的水平较对照组显著升高;联合免疫组较单独免疫组小鼠IFN-γ的水平升高更显著;单独、联合免疫组及对照空白质粒组小鼠在免疫前后脾细胞分泌抗原特异性细胞因子IL-4的水平无明显改变。
②体液免疫评价:单独及联合免疫组小鼠血清抗原特异性IgG抗体水平较对照空白质粒组显著升高;联合免疫组较单独免疫组小鼠IgG抗体水平升高更显著。
(2)免疫保护效能评价
①肺炎链球菌鼻咽部定植的保护:单独及联合免疫组小鼠鼻咽部肺炎链球菌数量与对照空白质粒及PBS组比较有所减少;联合免疫组较单独免疫组小鼠鼻咽部肺炎链球菌数量减少更显著。
②肺炎链球菌侵袭性腹腔攻击的保护:单独及联合免疫组小鼠中位生存时间与空白质粒及PBS对照组比较明显延长;联合免疫组较单独免疫组小鼠中位生存时间延长更显著。
2.新型减毒沙门菌为载体的肺炎链球菌多价DNA疫苗免疫原性及保护效能
(1)新型减毒沙门菌为载体的肺炎链球菌多价DNA疫苗的构建:成功构建真核互补质粒pcDNA3.1000,引进了穿梭质粒载体的必要元件真核启动子Pcmv ,多克隆位点MCS ,多聚腺苷酸尾BGHpA及原核复制子pUCori,同时去除了与病毒复制有关的SV40ori及SV40pA ,并以营养选择标志基因asd代替了原质粒载体上的氨苄抗性基因Amp及新霉素抗性基因Neo。进一步分子克隆技术成功构建新型减毒沙门菌为载体的肺炎链球菌多价DNA疫苗pcDNA3.1001x(编码psaA基因),pcDNA3.1002x(编码pspA’基因),转录水平和翻译水平成功检测到pspA’及psaA基因在哺乳动物BHK-21细胞的表达,新系统经传代培养25代,重组真核质粒pcDNA3.1001及pcDNA3.1002稳定性高达100%。
(2)免疫原性评价
①细胞免疫评价:新型减毒沙门菌为载体的DNA疫苗各免疫组小鼠脾细胞分泌LPS特异性细胞因子IFN-γ的水平升高,各组间无显著性差异;新型减毒沙门菌为载体的DNA疫苗组脾细胞分泌PspA及PsaA抗原特异性细胞因子IFN-γ的水平与裸质粒DNA疫苗组比较显著升高;单独、联合免疫组及对照空白质粒组小鼠免疫前后脾细胞分泌LPS、PspA及PsaA抗原特异性细胞因子IL-4的水平无明显改变,各组间亦无显著差异。
②体液免疫评价:新型减毒沙门菌为载体的DNA疫苗联合免疫组小鼠血清抗PsaA及PspA抗原特异性IgG抗体水平与裸质粒DNA疫苗组比较显著升高;所有单独及联合免疫组小鼠血清PsaA及PspA抗原特异性IgG抗体水平均显著高于对照空白质粒组;所有联合免疫组小鼠血清PsaA及PspA抗原特异性IgG抗体水平均较单独免疫组显著升高。
③黏膜免疫评价:新型减毒沙门菌为载体的DNA疫苗免疫组小鼠鼻咽部灌洗液中sIgG水平教空白质粒对照组显著升高,联合免疫组小鼠鼻咽部灌洗液中抗PsaA及PspA抗原特异性sIgG抗体水平较单独免疫组显著升高。
(3)免疫保护效能评价
①肺炎链球菌鼻咽部定植的保护:新型减毒沙门菌为载体的DNA疫苗免疫组与裸质粒DNA疫苗组比较小鼠鼻咽部肺炎链球菌数量减少更为显著;并且联合免疫组较单独免疫组具有更好的保护效能。
②肺炎链球菌侵袭性腹腔攻击的保护:新型减毒沙门菌为载体的DNA疫苗免疫组与裸质粒DNA疫苗组比较小鼠中位生存时间显著延长;并且联合免疫组较单独免疫组具有更好的保护效能。
3.肺炎链球菌新一代基因工程疫苗优势靶抗原PsaA、PspA在鼻咽部微环境共栖同种群细菌中的分子遗传学分析
(1)肺炎链球菌psaA基因的种群基因起源进化分析提示psaA基因在临床分离的轻链球菌群菌株中广泛存在,但是仅仅在肺炎链球菌中该基因是垂直遗传的,其它的轻链球菌群菌株中psaA基因可能来源于共生环境中基因的水平转移,并且推测这些转移的psaA基因之间的差异可能与BOX元件的参与及转移后发生的频繁的基因内重组现象相关。
(2)肺炎链球菌pspA基因N端抗原多样性区域基因组学分析提示本地区PspA蛋白家族分布趋势:PspA-Fam1-Clade2(31.0%)和PspA-Fam2-Clade3(47.6%)的菌株作为优势分布,与国外报道存在差异。
结论:
新型减毒沙门菌载体系统在保障重组质粒的稳定性同时避免了抗性基因及减毒株毒力回复的安全问题,将其应用于携带肺炎链球菌多价DNA疫苗,激发了机体更全面的全身性免疫应答,在肺炎链球菌的定植及侵袭性感染的动物模型中表现出了增强的保护效能,是一种具备良好应用前景的肺炎链球菌基因工程疫苗优化策略。肺炎链球菌基因工程疫苗的优势靶抗原psaA和pspA基因的抗原遗传背景分析证实了这两种抗原在肺炎链球菌的抗原特异性。但pspA基因在本地区肺炎链球菌株中的抗原多样性现象提示:在进一步的肺炎链球菌基因工程疫苗靶抗原优化策略中应包含PspA优势家族抗原表位才能更全面的覆盖本地区的临床致病肺炎链球菌菌株感染。
Background and Objective
Streptococcus pneumoniae (S. pneumoniae) is the main pathogen of community- acquired pneumonia, otitis media. There are more than one hundred million cases of children under the age of 5 died of S. pneumoniae infection every year worldover. At present, the more and more serious drug- resistances of S. pneumoniae have became a major global public health problem and endangered the treatment of S. pneumoniae. Application of vaccines for combating pneumococcal infections is the only way proposed by World Health Organization, which should be help to reduce the spread of antibiotic resistance strains and alleviat the stress of antibiotics and protect high-risk groups.
More and more domestic and foreign clinical studies found the shortcomings of current pneumococcal vaccines (23-valent capsular polysaccharide vaccine and protein - polysaccharide conjugate vaccine), which are the serotype-dependent existence, the high cost, and non-reducing S. pneumoniae nasopharyngeal colonization. The study of new genome-based vaccines of S. pneumoniae has far-reaching social and economic benefits.
The large numbers of S. pneumoniae virulence proteins play the important and different roles at different pathogenic stages of S. pneumoniae infections. There is growing interest in S. pneumoniae DNA vaccine researches as the third vaccines, not only because of its low production cost, the non-serotype-dependent existence, but also the advantage Th1 immune responses.
The choices of target antigens and edge vector systems and ideal inoculations of vaccines are the most important questiones. Preliminary studies by our group have confirmed that the combination of pneumococcal surface protein A (PspA ) and pneumococcal surface adhesion A (PsaA) were the most forms. Since pneumococcal nasopharyngeal colonization is the native mucosal infective route that leads to invasive infection diseases, a DNA vaccine with adhesion protein genes against S. pneumoniae nasopharyngeal colonization should ideally produce protective secretory IgA antibodies in the respiratory mucosa as well as systemic IgG antibodies, in addition to Th1 cellular immune responses, which is hoped to reduce the transmission of antibiotic-resistant strains and prevent further pneumococcal invasive diseases. But a major drawback of the DNA vaccines is its poor mucosal protective efficiency in the nasopharynx, which is inherent in the way that vaccines are currently delivered. An approach that incorporates the advantages of both genetic immunization and mucosal immunization of S. pneumoniae DNA vaccine is thus required.
Therefore, this study“study on genome-based Streptococcus pneumoniae vaccines”was as the following works: First, S. pneumoniae virulence protein multivalent DNA vaccines were constructed based pspA’(N-terminal fragment of Streptococcus pneumoniae R6) and psaA, and the immunogenicity and protection effects were evaluated based the animal models of S. pneumoniae nasopharyngeal carriage and intraperitoneal challenges through the way of naked plasmid intramuscular vaccination. The second, we constructed a recombinant salmonella-based balanced-lethal host-vector system of the avirulent Salmonella strainχ4550(Asd-) with eukaryotic expression Asd+ vectors pcDNA3.1001 and pcDNA3.1002 expressing psaA gene or pspA’gene. The stability, immunogenicity (especially mucosal immunity) and the protective abilities against nasopharyngeal carriage S. pneumoniae and invasive diseases were evaluated in mice immunized with the antigen delivery systems with combination of PspA and PsaA for intranasal vaccinations, comparing to S. pneumoniae virulence protein multivalent DNA vaccines. The last, we study preliminary the comparative genomics of dominant antigens by analyzing the sequence characteristics of dominant antigen proteins from molecular level and their possible functional differences, which was for learning more about the possible antigen diversity of dominant antigen proteins on the basis of molecular genetics.
Thus, the results provide the theoretical foundation and laboratory basis for the development of new generation Streptococcus pneumoniae vaccines.
Methods
1. The pspA’(N-terminal fragment of Streptococcus pneumoniae R6) and psaA genes were cloned into the eukaryotic expression vector pcDNA3.1(+) by molecular cloning technology. The recombinant vectors PsaA-pcDNA3.1 and PspA’-pcDNA3.1 were transfected into BHK-21 cells, and the expression of PsaA and PspA’proteins was detected with RT-PCR and Western- blot. The three formats of recombinant plasmids (PspA’-pcDNA3.1, PsaA-pcDNA3.1 and PsaA-pcDNA3.1 + PspA’-pcDNA3.1) were used to inject intramuscularly BALB/c mice. The levels of IFN-γ, IL-4 and antibodies against PspA’, PsaA were checked. The nasopharyngeal colony count of Streptococcus pneumoniae D39 strain and the live time of intraperitoneal infection mice by Streptococcus pneumoniae D39 strain were analysis in BALB/c models.
2. Oral DNA vaccines encoding psaA and pspA’with Salmonella-based balanced-lethal host- eukaryotic vector system as carriers were reconstructed using molecular biology techniques. And the stability, immunogenicity (especially mucosal immunity) and the protective abilities against nasopharyngeal carriage S. pneumoniae and invasive diseases were evaluated in mice models.
⑴To construct the Asd+ eukaryotic vector pcDNA3.1000 contained in the Salmonella-based balanced-lethal host-eukaryotic vector system, we reconstructed the eukaryotic vector pcDNA3.1 (+), which is under transcriptional control of the human CMV promoter. First, a 2.6 kb fragment of SV40ori- Neor-SV40pA-pUCori and 1.9 kb fragment of Pcmv-MCS- BGHpA-f1ori were retried through digesting pcDNA3.1(+) with BspHI and XmnI restriction enzymes. Then the pUCori fragment was obtained with SalI restriction enzymes through digesting the 2.6 kb fragment of SV40ori- Neor-SV40pA-pUCori, while the 1.07 kb asd gene fragment was amplified from Streptococcus mutans UA159 by PCR with an N-terminal XhoI site and a C-terminal SalI site. The reaction was carried out. Thus, the 1.07 kb fragment of the asd gene was obtained, and then the fragments of pUCori, asd and Pcmv-MCS-BGHpA-f1ori were connected with T4 DNA ligase. Thus the fragments of Neor and AMP gene sequences were deleted from pcDNA3.1(+), and the asd gene sequence was obtained, and then the recombinant vector pcDNA3.1000 (Asd+) was amplified intoχ4550(Asd-).
⑵The new oral DNA vaccines encoding the two most promising candidates antigens psaA and pspA’genes with Salmonella-based balanced-lethal host-vector system as carriers were constructed using molecular cloning technology. And the recombinant vectors pcDNA3.1001 and pcDNA3.1002 were transfected into BHK-21 cells, and the expression of PsaA and PspA’proteins was detected with RT-PCR and Western- blot. The three formats of oral DNA vaccines (pcDNA3.1001x, pcDNA3.1002x and pcDNA3.1001x +pcDNA3.1002x) were used to orally deliver to BALB/c mice, with the vector pcDNA3.1000x and PBS as controls. Comparing to vector DNA vaccines (pcDNA3.101, PsaA-pcDNA3.102 and pcDNA3.101 +pcDNA3.102), the levels of IFN-γ, IL-4 and antibodies against PspA’, PsaA were checked. The nasopharyngeal colony count of Streptococcus pneumoniae D39 strain and the live time of intraperitoneal infection mice by Streptococcus pneumoniae D39 strain were analysis in BALB/c models.
3. Methods of molecular genetics and immunology technology were used for further analyzing the comparative genomics and the diversity of protein antigens as the third generation Streptococcus pneumoniae vaccine antigen proteins.
⑴Presence of psaA gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. The characters of these psaA genes sequences were compared, and phylogenies basing on the sequences of housekeeping genes and PsaA genes were producted for analyzing the possible genetic mechanisms of PsaA gene in streptococcus mitis group.
⑵Presence of pspA’gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. Phylogenies basing on the sequences of pspA’genes from Streptococcus pneumoniae strains were typed for the PspA families and clades.
Results
1. The immunogenicity and protection effects in mice immunized intramuscularly with naked plasmid Streptococcus pneumoniae multivalent DNA vaccines.
⑴RT-PCR and Western- blot analysis of total cell extracts showed successful transcription and expression of PspA’-pcDNA3.1 and PsaA-pcDNA3.1 in BHK-21 cells.
⑵Immune responses in mice immunized with naked plasmid Streptococcus pneumoniae DNA vaccines PspA’-pcDNA3.1 and PsaA-pcDNA3.1.
①Cell immune responses. PsaA and PspA’antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with PspA’-pcDNA3.1 and PsaA-pcDNA3.1 alone or combined were all significantly higher on 21th day after last immunization than that on day 0, and significantly higher than the control plasmid group; PsaA and PspA 'antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with PspA’-pcDNA3.1 + PsaA-pcDNA3.1 were significantly higher than that with PspA’-pcDNA3.1 or PsaA-pcDNA3.1 alone on 21th day after last immunization; PsaA and PspA 'antigen-specific cytokine IL-4 in spleen cells supernatant of mice group immunized with PspA’-pcDNA3.1 and PsaA-pcDNA3.1, alone or combined, and plamids contral group were all low on day 0 or on 21th day after last immunization, and there had no significantly divergence between on day 0 with on 21th day after last immunization,or among all groups.
②Humoral immune responses. PsaA and PspA’antigen-specific IgG antibodies in serum of mice immunized with PspA’-pcDNA3.1 and PsaA-pcDNA3.1 alone or combined were all significantly higher on 28th day after last immunization; PsaA and PspA’antigen-specific IgG antibodies in serum of mice immunized with PspA’-pcDNA3.1+ PsaA-pcDNA3.1 was higher than that with PspA’-pcDNA3.1 or PsaA-pcDNA3.1 alone.
⑶Evaluation of levels of carriage and protective immunity in mice immunized intramuscularly with naked plasmid S. pneumoniae multivalent DNA vaccines PspA’-pcDNA3.1 and PsaA-pcDNA3.1.
①The protection against S. pneumoniae D39 nasopharyngeal colonization. The result showed fewer pneumococci were recovered from mice immunized with naked plasmid S. pneumoniae multivalent DNA vaccines PspA’-pcDNA3.1 and PsaA-pcDNA3.1, alone or combined, than the contral plasmid and PBS groups. And the effect of immunization on carriage with mixed DNA vaccine plasmids was highly significant versus with one of them alone.
②The protection against S. pneumoniae D39 invasive infection. In the intraperitoneal-challenge experiments, we observed the corresponding result that mice immunizaed with the mixed DNA vaccines plasmids PspA’-pcDNA3.1+ PsaA-pcDNA3.1 had significantly longer median survival times than the group that received alone with one of them.
2. The immunogenicity and protection effects in mice immunized intramuscularly with oral S. pneumoniae multivalent DNA vaccines with the new carrier system.
⑴RT-PCR and Western- blot analysis of total cell extracts showed successful transcription and expression of pcDNA3.1001 and pcDNA3.1002 in BHK-21 cells. All of these plasmids(pcDNA3.1000, pcDNA3.1001, pcDNA3.1002) were 100% stably maintained for 25 generations in S. typhimurium (Asd-) hosts grown in the presence or absence of DAP.
⑵Immune responses in mice immunized with oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x.
①Cell immune responses. LPS antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with pcDNA3.1000x, pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x+pcDNA3.1002x were all significantly higher on 21th day after last immunization than that on day 0, but there had no significantly difference among all groups; PsaA or PspA antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with pcDNA3.1000x, pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x + pcDNA3.1002x were all significantly higher on 21th day after last immunization than that on day 0, and PsaA or PspA antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with oral DNA vaccines(pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x+pcDNA3.1002x ) were significantly higher than DNA vaccine plamids(pcDNA3.101, pcDNA3.102 or pcDNA3.101+pcDNA3.102); PsaA and PspA’antigen-specific cytokine IL-4 in spleen cells supernatant of all mice group immunized were all low on day 0 or on 21th day after last immunization, and there had no significantly divergence between on day 0 with on 21th day after last immunization,or among all groups.
②Humoral immune responses. The antibody responses to Salmonella LPS and to the foreign antigen PspA’or PsaA in the sera and the nasal wash of the immunized mice were measured. Responses reached a maximal anti-LPS, -PsaA, and–PspA’IgG levels on 28th day after last immunization and all IgG antibody responses in vaccine groups were significantly stronger than the control pcDNA3.1 (+) and pcDNA3.1000x groups. Anti-LPS IgG antibody levels were similar in the mice of groups immunized by all oral DNA vaccines. IgG antibody levels against foreign antigens (PspA’or PsaA) in the mice of groups immunized by the oral multiantigen DNA vaccines ( pcDNA3.1001x+ pcDNA3.1002x)were significantly higher than that by the multiantigen DNA vaccine plasmids (i.m) (pcDNA3.101+ pcDNA3.102). IgG antibody levels against PspA’or PsaA in the mice of groups immunized by the multiantigen DNA vaccines (pcDNA3.1001x+pcDNA3.1002x or pcDNA3.101+ pcDNA3.102 )were significantly higher than that by the univalent DNA vaccines encoding either one of pspA’and psaA (including pcDNA3.1001x, pcDNA3.1002x and pcDNA3.101, pcDNA3.102).
③Mucosal immune responses. Secretory IgA antibodies to PspA’and PsaA were detected in the nasal washes of mice immunized by oral DNA vaccines, and negligible secretory IgA elicited by the DNA vaccine plasmids (i.m.) group. On 28th day after last immunization, the antibody leves reached peak, and higher IgA antibody levels against either PspA’or PsaA were observed in the groups of mice immunized with oral multiantigen DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) than in mice immunized with one of them alone (pcDNA3.1001x, pcDNA3.1002x) and the control group pcDNA3.1000x.
⑶Evaluation of levels of carriage and protective immunity in mice immunized intramuscularly with oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x.
①The protection against S. pneumoniae D39 nasopharyngeal colonization. The result showed fewer pneumococci were recovered from mice immunized with oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) than that immunited with the DNA vaccines (pcDNA3.101+ pcDNA3.102). The effect of immunization on carriage with mixed oral DNA vaccine pcDNA3.1001x +pcDNA3.1002x was highly significant versus with one of them alone pcDNA3.1001x or pcDNA3.1002x.
②The protection against S. pneumoniae D39 invasive infection. In the intraperitoneal-challenge experiments, we observed the corresponding result that mice immunizaed with oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) had significantly longer median survival times than that immunited with the DNA vaccines (pcDNA3.101+ pcDNA3.102). And the mice immunizaed with mixed oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) had significantly longer median survival times than that immunited with one of them alone.
3. High frequency events of HCG and recombination support the prevalent presence of PsaA gene in streptococcus mitis group. These species may acquire transforming PsaA gene from the other species of streptococcus mitis group living in the same ecological niche, and maybe involved in promote the adhesion behavior and subsequent oropharyngeal colonization behavior.
4. PspA gene diversity of the region N terminal antigen genomic analysis suggested that the trend of regional distribution of PspA protein family: PspA-Fam1-Clade2 (31.0%) and PspA-Fam2-Clade3 (47.6%) as the predominant strains, and differences in foreign reports.
Conclusion
Successfully, oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x induced significant immune responses versus the DNA vaccine plasmids pcDNA3.101 and pcDNA3.102 intramuscular injected (i.m.), containing mucosa, cellular and humoral immune responses, and enhanced protection against nasopharyngeal colonization and protection against the intraperitoneal challenge. The studies also confirmed the cooperating, additive protection with multiantigen DNA vaccines against S. pneumoniae nasopharyngeal colonization and infection. The results provided the potential of using the salmonella-based balanced-lethal host-eukaryotic vector system as carriers to deliver S. pneumoniae protective antigens.
PsaA and PspA are the best forms of dominant antigen candidates of the new Streptococcus pneumonia generation vaccines. PsaA gene is vertical inheritance in Streptococcus pneumoniae, which may be aquired by other symbiotic streptococcus through horizontal gene transfer and recombinant gene manner. PspA gene has the diversity, and the target antigens should also include the clades components of Fam1 and Fam2 with high protective and strong cross-reaction against a wide range of clinical pathogenic strains of Streptococcus pneumoniae.
肺炎链球菌是社区获得性感染最常见的致病菌,全球每年有超过100万儿童死于肺炎链球菌感染性疾病。目前肺炎链球菌对抗生素多重耐药情况日趋严重,使肺炎链球菌感染的治疗面临重大挑战,WHO提出应用疫苗预防肺炎链球菌感染是减少耐药菌传播、缓解抗生素耐药压力,保护高危人群的唯一方法。
国内外越来越多的临床研究发现目前应用的肺炎链球菌23价荚膜多糖疫苗和7价蛋白-多糖结合疫苗存在血清型依赖,成本昂贵,不降低肺炎链球菌鼻咽部携带等不足。研发新一代非血清型依赖的安全、便携、廉价的新型肺炎链球菌疫苗具有巨大的经济效益和深远的社会价值。
肺炎链球菌作为条件致病菌,多种毒力因子独立或协同的参与了其粘附、定植、迁移、侵袭性致病的全过程。基于肺炎链球菌相关毒力蛋白的DNA疫苗具有非血清型依赖、覆盖率广、造价低廉等优势,成为第三代肺炎链球菌疫苗的研究热点。靶抗原的选择,优势载体系统的应用和理想的接种途径是新型肺炎链球菌DNA疫苗研究的关键环节。本课题组前期研究已经证实了肺炎链球菌表面蛋白A(pneumococcal surface protein A,PspA)及肺炎链球菌黏附素A(pneumococcal surface adhesion A ,PsaA)的联合是最优势的候选抗原组合形式,但是传统的DNA疫苗免疫途径并不具备激发鼻咽部特异性sIgA抗体保护的能力,而且免疫原性较弱。鉴于鼻咽部定植是肺炎链球菌感染的首要步骤,研究如何优化DNA疫苗的载体系统,选择理想的免疫途径进而增强肺炎链球菌DNA疫苗的免疫效能,尤其是增加其鼻咽部粘膜免疫保护效能具有重要意义。
因此,本课题围绕“肺炎链球菌基因工程疫苗的实验研究”,进行了以下三部分研究工作:(1)第一部分:基于优势靶抗原组合(pspA+psaA基因),构建肺炎链球菌多价DNA疫苗。复制肺炎链球菌鼻咽部携带及腹腔攻击动物模型,裸质粒肌肉注射方式免疫接种,评价其免疫原性及保护效能;(2)第二部分:基于载体-宿主致死平衡原理,设计改造现有减毒沙门菌为宿主菌的沙门菌-原核质粒平衡致死系统,将其携带的原核质粒改造为非抗性基因筛选的真核质粒,构建新型减毒沙门菌为载体的多价肺炎链球菌DNA基因工程疫苗,复制肺炎链球菌鼻咽部携带及腹腔攻击动物模型,经口服途径免疫接种,评价其稳定性、免疫原性及免疫保护效能;(3)第三部分:进一步利用基因组学及分子遗传学技术针对优势靶抗原进行分子进化及比较基因组学的初步研究。旨在为肺炎链球菌新一代基因工程疫苗的研发提供理论基础和实验室依据。方法:
1.分子克隆技术构建PsaA和PspA’(PspA的N端抗原表位所在区域基因片段)的重组真核表达质粒PsaA-pcDNA3.1和PspA’-pcDNA3.1。两种重组质粒DNA疫苗PsaA-pcDNA3.1和PspA’-pcDNA3.1裸质粒单独或联合肌肉注射免疫小鼠,ELISA检测免疫小鼠脾细胞分泌细胞因子及血清中特异性抗体IgG水平;复制小鼠肺炎链球菌D39株鼻咽部携带和腹腔侵袭性感染模型,观察免疫小鼠鼻咽部肺炎链球菌携带改变和腹腔攻击小鼠21天生存情况。
2.新型减毒沙门菌为载体的多价肺炎链球菌DNA疫苗构建及其稳定性、免疫效能检测。
(1)肺炎链球菌DNA疫苗的新型减毒沙门菌载体系统构建:基于pcDNA3.1(+),应用BspHI、XmnⅠ双酶切获得片段SV40ori-Neor-SV40Pa-pUCori和Pcmv-MCS-BGHpA-f1ori,再用SalⅠ酶切SV40ori-Neor-SV40pA-pUCori获得片段pUCori;将酶切获得的pUCori、Pcmv-MCS-BGHpA-f1ori两个片段及变形链球菌asd基因PCR扩增产物进行连接,构建新互补质粒pcDNA3.1000,并最终转化入宿主菌沙门菌χ4550中(pcDNA3.1000x)。
(2)分子克隆技术构建新型减毒沙门菌为载体的肺炎链球菌DNA疫苗pcDNA3.1001x(编码pspA’基因)和pcDNA3.1002x(编码psaA基因),并检测重组质粒在哺乳动物细胞BHK-21中的表达(RT-PCR及Western-blot)及质粒稳定性。新型减毒沙门菌为载体的肺炎链球菌DNA疫苗pcDNA3.1001x和pcDNA3.1002x单独或联合口服免疫小鼠,ELISA检测免疫小鼠脾细胞分泌的细胞因子、血清中特异性抗体IgG及鼻咽部灌洗液中sIgG水平。复制小鼠肺炎链球菌D39株鼻咽部携带模型和腹腔侵袭性感染模型,观察免疫小鼠鼻咽部肺炎链球菌D39菌落计数改变和腹腔攻击小鼠21天生存情况,以评价新型减毒沙门菌为载体的多价肺炎链球菌DNA疫苗的免疫原性及保护作用优势。
3.采用分子遗传学技术进一步分析肺炎链球菌基因工程疫苗优势抗原蛋白的分子进化特点及抗原多样性。
(1)采集上呼吸道共栖环境中的临床收集轻链球菌家族菌株(包含肺炎链球菌、轻链球菌、口腔链球菌、血链球菌、婴儿链球菌),PCR技术探查psaA基因分布情况。基于扩增的psaA基因进行氨基酸序列比对及系统发生树构建,分析psaA的序列特点及在轻链球菌家族中可能的遗传机制。
(2)采集本地区临床致病肺炎链球菌感染菌株,应用PspA蛋白家族分型特异性引物PCR及生物信息学技术鉴定本地区临床自然感染肺炎链球菌株PspA家族分布特点;进一步应用分子遗传学技术构建pspA基因多家族的系统发生树,分析肺炎链球菌PspA作为新一代肺炎链球菌基因工程疫苗优势候选抗原应包含的优势家族成分。
结果:
1.肺炎链球菌多价DNA疫苗的构建:成功扩增pspA基因包含抗原表位的N端片段及psaA基因目标片段,分别克隆入真核载体pcDNA3.1(+),构建了PspA’-pcDNA3.1和PsaA-pcDNA3.1两种肺炎链球菌毒力蛋白重组真核质粒,并从转录水平和翻译水平检测了这两种重组质粒编码的psaA及pspA’基因在哺乳动物细胞的成功表达。
2.肺炎链球菌多价DNA疫苗的免疫原性及保护效能
(1)免疫原性评价
①细胞免疫评价:单独及联合免疫组小鼠脾细胞分泌的抗原特异性细胞因子IFN-γ的水平较对照组显著升高;联合免疫组较单独免疫组小鼠IFN-γ的水平升高更显著;单独、联合免疫组及对照空白质粒组小鼠在免疫前后脾细胞分泌抗原特异性细胞因子IL-4的水平无明显改变。
②体液免疫评价:单独及联合免疫组小鼠血清抗原特异性IgG抗体水平较对照空白质粒组显著升高;联合免疫组较单独免疫组小鼠IgG抗体水平升高更显著。
(2)免疫保护效能评价
①肺炎链球菌鼻咽部定植的保护:单独及联合免疫组小鼠鼻咽部肺炎链球菌数量与对照空白质粒及PBS组比较有所减少;联合免疫组较单独免疫组小鼠鼻咽部肺炎链球菌数量减少更显著。
②肺炎链球菌侵袭性腹腔攻击的保护:单独及联合免疫组小鼠中位生存时间与空白质粒及PBS对照组比较明显延长;联合免疫组较单独免疫组小鼠中位生存时间延长更显著。
2.新型减毒沙门菌为载体的肺炎链球菌多价DNA疫苗免疫原性及保护效能
(1)新型减毒沙门菌为载体的肺炎链球菌多价DNA疫苗的构建:成功构建真核互补质粒pcDNA3.1000,引进了穿梭质粒载体的必要元件真核启动子Pcmv ,多克隆位点MCS ,多聚腺苷酸尾BGHpA及原核复制子pUCori,同时去除了与病毒复制有关的SV40ori及SV40pA ,并以营养选择标志基因asd代替了原质粒载体上的氨苄抗性基因Amp及新霉素抗性基因Neo。进一步分子克隆技术成功构建新型减毒沙门菌为载体的肺炎链球菌多价DNA疫苗pcDNA3.1001x(编码psaA基因),pcDNA3.1002x(编码pspA’基因),转录水平和翻译水平成功检测到pspA’及psaA基因在哺乳动物BHK-21细胞的表达,新系统经传代培养25代,重组真核质粒pcDNA3.1001及pcDNA3.1002稳定性高达100%。
(2)免疫原性评价
①细胞免疫评价:新型减毒沙门菌为载体的DNA疫苗各免疫组小鼠脾细胞分泌LPS特异性细胞因子IFN-γ的水平升高,各组间无显著性差异;新型减毒沙门菌为载体的DNA疫苗组脾细胞分泌PspA及PsaA抗原特异性细胞因子IFN-γ的水平与裸质粒DNA疫苗组比较显著升高;单独、联合免疫组及对照空白质粒组小鼠免疫前后脾细胞分泌LPS、PspA及PsaA抗原特异性细胞因子IL-4的水平无明显改变,各组间亦无显著差异。
②体液免疫评价:新型减毒沙门菌为载体的DNA疫苗联合免疫组小鼠血清抗PsaA及PspA抗原特异性IgG抗体水平与裸质粒DNA疫苗组比较显著升高;所有单独及联合免疫组小鼠血清PsaA及PspA抗原特异性IgG抗体水平均显著高于对照空白质粒组;所有联合免疫组小鼠血清PsaA及PspA抗原特异性IgG抗体水平均较单独免疫组显著升高。
③黏膜免疫评价:新型减毒沙门菌为载体的DNA疫苗免疫组小鼠鼻咽部灌洗液中sIgG水平教空白质粒对照组显著升高,联合免疫组小鼠鼻咽部灌洗液中抗PsaA及PspA抗原特异性sIgG抗体水平较单独免疫组显著升高。
(3)免疫保护效能评价
①肺炎链球菌鼻咽部定植的保护:新型减毒沙门菌为载体的DNA疫苗免疫组与裸质粒DNA疫苗组比较小鼠鼻咽部肺炎链球菌数量减少更为显著;并且联合免疫组较单独免疫组具有更好的保护效能。
②肺炎链球菌侵袭性腹腔攻击的保护:新型减毒沙门菌为载体的DNA疫苗免疫组与裸质粒DNA疫苗组比较小鼠中位生存时间显著延长;并且联合免疫组较单独免疫组具有更好的保护效能。
3.肺炎链球菌新一代基因工程疫苗优势靶抗原PsaA、PspA在鼻咽部微环境共栖同种群细菌中的分子遗传学分析
(1)肺炎链球菌psaA基因的种群基因起源进化分析提示psaA基因在临床分离的轻链球菌群菌株中广泛存在,但是仅仅在肺炎链球菌中该基因是垂直遗传的,其它的轻链球菌群菌株中psaA基因可能来源于共生环境中基因的水平转移,并且推测这些转移的psaA基因之间的差异可能与BOX元件的参与及转移后发生的频繁的基因内重组现象相关。
(2)肺炎链球菌pspA基因N端抗原多样性区域基因组学分析提示本地区PspA蛋白家族分布趋势:PspA-Fam1-Clade2(31.0%)和PspA-Fam2-Clade3(47.6%)的菌株作为优势分布,与国外报道存在差异。
结论:
新型减毒沙门菌载体系统在保障重组质粒的稳定性同时避免了抗性基因及减毒株毒力回复的安全问题,将其应用于携带肺炎链球菌多价DNA疫苗,激发了机体更全面的全身性免疫应答,在肺炎链球菌的定植及侵袭性感染的动物模型中表现出了增强的保护效能,是一种具备良好应用前景的肺炎链球菌基因工程疫苗优化策略。肺炎链球菌基因工程疫苗的优势靶抗原psaA和pspA基因的抗原遗传背景分析证实了这两种抗原在肺炎链球菌的抗原特异性。但pspA基因在本地区肺炎链球菌株中的抗原多样性现象提示:在进一步的肺炎链球菌基因工程疫苗靶抗原优化策略中应包含PspA优势家族抗原表位才能更全面的覆盖本地区的临床致病肺炎链球菌菌株感染。
Background and Objective
Streptococcus pneumoniae (S. pneumoniae) is the main pathogen of community- acquired pneumonia, otitis media. There are more than one hundred million cases of children under the age of 5 died of S. pneumoniae infection every year worldover. At present, the more and more serious drug- resistances of S. pneumoniae have became a major global public health problem and endangered the treatment of S. pneumoniae. Application of vaccines for combating pneumococcal infections is the only way proposed by World Health Organization, which should be help to reduce the spread of antibiotic resistance strains and alleviat the stress of antibiotics and protect high-risk groups.
More and more domestic and foreign clinical studies found the shortcomings of current pneumococcal vaccines (23-valent capsular polysaccharide vaccine and protein - polysaccharide conjugate vaccine), which are the serotype-dependent existence, the high cost, and non-reducing S. pneumoniae nasopharyngeal colonization. The study of new genome-based vaccines of S. pneumoniae has far-reaching social and economic benefits.
The large numbers of S. pneumoniae virulence proteins play the important and different roles at different pathogenic stages of S. pneumoniae infections. There is growing interest in S. pneumoniae DNA vaccine researches as the third vaccines, not only because of its low production cost, the non-serotype-dependent existence, but also the advantage Th1 immune responses.
The choices of target antigens and edge vector systems and ideal inoculations of vaccines are the most important questiones. Preliminary studies by our group have confirmed that the combination of pneumococcal surface protein A (PspA ) and pneumococcal surface adhesion A (PsaA) were the most forms. Since pneumococcal nasopharyngeal colonization is the native mucosal infective route that leads to invasive infection diseases, a DNA vaccine with adhesion protein genes against S. pneumoniae nasopharyngeal colonization should ideally produce protective secretory IgA antibodies in the respiratory mucosa as well as systemic IgG antibodies, in addition to Th1 cellular immune responses, which is hoped to reduce the transmission of antibiotic-resistant strains and prevent further pneumococcal invasive diseases. But a major drawback of the DNA vaccines is its poor mucosal protective efficiency in the nasopharynx, which is inherent in the way that vaccines are currently delivered. An approach that incorporates the advantages of both genetic immunization and mucosal immunization of S. pneumoniae DNA vaccine is thus required.
Therefore, this study“study on genome-based Streptococcus pneumoniae vaccines”was as the following works: First, S. pneumoniae virulence protein multivalent DNA vaccines were constructed based pspA’(N-terminal fragment of Streptococcus pneumoniae R6) and psaA, and the immunogenicity and protection effects were evaluated based the animal models of S. pneumoniae nasopharyngeal carriage and intraperitoneal challenges through the way of naked plasmid intramuscular vaccination. The second, we constructed a recombinant salmonella-based balanced-lethal host-vector system of the avirulent Salmonella strainχ4550(Asd-) with eukaryotic expression Asd+ vectors pcDNA3.1001 and pcDNA3.1002 expressing psaA gene or pspA’gene. The stability, immunogenicity (especially mucosal immunity) and the protective abilities against nasopharyngeal carriage S. pneumoniae and invasive diseases were evaluated in mice immunized with the antigen delivery systems with combination of PspA and PsaA for intranasal vaccinations, comparing to S. pneumoniae virulence protein multivalent DNA vaccines. The last, we study preliminary the comparative genomics of dominant antigens by analyzing the sequence characteristics of dominant antigen proteins from molecular level and their possible functional differences, which was for learning more about the possible antigen diversity of dominant antigen proteins on the basis of molecular genetics.
Thus, the results provide the theoretical foundation and laboratory basis for the development of new generation Streptococcus pneumoniae vaccines.
Methods
1. The pspA’(N-terminal fragment of Streptococcus pneumoniae R6) and psaA genes were cloned into the eukaryotic expression vector pcDNA3.1(+) by molecular cloning technology. The recombinant vectors PsaA-pcDNA3.1 and PspA’-pcDNA3.1 were transfected into BHK-21 cells, and the expression of PsaA and PspA’proteins was detected with RT-PCR and Western- blot. The three formats of recombinant plasmids (PspA’-pcDNA3.1, PsaA-pcDNA3.1 and PsaA-pcDNA3.1 + PspA’-pcDNA3.1) were used to inject intramuscularly BALB/c mice. The levels of IFN-γ, IL-4 and antibodies against PspA’, PsaA were checked. The nasopharyngeal colony count of Streptococcus pneumoniae D39 strain and the live time of intraperitoneal infection mice by Streptococcus pneumoniae D39 strain were analysis in BALB/c models.
2. Oral DNA vaccines encoding psaA and pspA’with Salmonella-based balanced-lethal host- eukaryotic vector system as carriers were reconstructed using molecular biology techniques. And the stability, immunogenicity (especially mucosal immunity) and the protective abilities against nasopharyngeal carriage S. pneumoniae and invasive diseases were evaluated in mice models.
⑴To construct the Asd+ eukaryotic vector pcDNA3.1000 contained in the Salmonella-based balanced-lethal host-eukaryotic vector system, we reconstructed the eukaryotic vector pcDNA3.1 (+), which is under transcriptional control of the human CMV promoter. First, a 2.6 kb fragment of SV40ori- Neor-SV40pA-pUCori and 1.9 kb fragment of Pcmv-MCS- BGHpA-f1ori were retried through digesting pcDNA3.1(+) with BspHI and XmnI restriction enzymes. Then the pUCori fragment was obtained with SalI restriction enzymes through digesting the 2.6 kb fragment of SV40ori- Neor-SV40pA-pUCori, while the 1.07 kb asd gene fragment was amplified from Streptococcus mutans UA159 by PCR with an N-terminal XhoI site and a C-terminal SalI site. The reaction was carried out. Thus, the 1.07 kb fragment of the asd gene was obtained, and then the fragments of pUCori, asd and Pcmv-MCS-BGHpA-f1ori were connected with T4 DNA ligase. Thus the fragments of Neor and AMP gene sequences were deleted from pcDNA3.1(+), and the asd gene sequence was obtained, and then the recombinant vector pcDNA3.1000 (Asd+) was amplified intoχ4550(Asd-).
⑵The new oral DNA vaccines encoding the two most promising candidates antigens psaA and pspA’genes with Salmonella-based balanced-lethal host-vector system as carriers were constructed using molecular cloning technology. And the recombinant vectors pcDNA3.1001 and pcDNA3.1002 were transfected into BHK-21 cells, and the expression of PsaA and PspA’proteins was detected with RT-PCR and Western- blot. The three formats of oral DNA vaccines (pcDNA3.1001x, pcDNA3.1002x and pcDNA3.1001x +pcDNA3.1002x) were used to orally deliver to BALB/c mice, with the vector pcDNA3.1000x and PBS as controls. Comparing to vector DNA vaccines (pcDNA3.101, PsaA-pcDNA3.102 and pcDNA3.101 +pcDNA3.102), the levels of IFN-γ, IL-4 and antibodies against PspA’, PsaA were checked. The nasopharyngeal colony count of Streptococcus pneumoniae D39 strain and the live time of intraperitoneal infection mice by Streptococcus pneumoniae D39 strain were analysis in BALB/c models.
3. Methods of molecular genetics and immunology technology were used for further analyzing the comparative genomics and the diversity of protein antigens as the third generation Streptococcus pneumoniae vaccine antigen proteins.
⑴Presence of psaA gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. The characters of these psaA genes sequences were compared, and phylogenies basing on the sequences of housekeeping genes and PsaA genes were producted for analyzing the possible genetic mechanisms of PsaA gene in streptococcus mitis group.
⑵Presence of pspA’gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. Phylogenies basing on the sequences of pspA’genes from Streptococcus pneumoniae strains were typed for the PspA families and clades.
Results
1. The immunogenicity and protection effects in mice immunized intramuscularly with naked plasmid Streptococcus pneumoniae multivalent DNA vaccines.
⑴RT-PCR and Western- blot analysis of total cell extracts showed successful transcription and expression of PspA’-pcDNA3.1 and PsaA-pcDNA3.1 in BHK-21 cells.
⑵Immune responses in mice immunized with naked plasmid Streptococcus pneumoniae DNA vaccines PspA’-pcDNA3.1 and PsaA-pcDNA3.1.
①Cell immune responses. PsaA and PspA’antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with PspA’-pcDNA3.1 and PsaA-pcDNA3.1 alone or combined were all significantly higher on 21th day after last immunization than that on day 0, and significantly higher than the control plasmid group; PsaA and PspA 'antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with PspA’-pcDNA3.1 + PsaA-pcDNA3.1 were significantly higher than that with PspA’-pcDNA3.1 or PsaA-pcDNA3.1 alone on 21th day after last immunization; PsaA and PspA 'antigen-specific cytokine IL-4 in spleen cells supernatant of mice group immunized with PspA’-pcDNA3.1 and PsaA-pcDNA3.1, alone or combined, and plamids contral group were all low on day 0 or on 21th day after last immunization, and there had no significantly divergence between on day 0 with on 21th day after last immunization,or among all groups.
②Humoral immune responses. PsaA and PspA’antigen-specific IgG antibodies in serum of mice immunized with PspA’-pcDNA3.1 and PsaA-pcDNA3.1 alone or combined were all significantly higher on 28th day after last immunization; PsaA and PspA’antigen-specific IgG antibodies in serum of mice immunized with PspA’-pcDNA3.1+ PsaA-pcDNA3.1 was higher than that with PspA’-pcDNA3.1 or PsaA-pcDNA3.1 alone.
⑶Evaluation of levels of carriage and protective immunity in mice immunized intramuscularly with naked plasmid S. pneumoniae multivalent DNA vaccines PspA’-pcDNA3.1 and PsaA-pcDNA3.1.
①The protection against S. pneumoniae D39 nasopharyngeal colonization. The result showed fewer pneumococci were recovered from mice immunized with naked plasmid S. pneumoniae multivalent DNA vaccines PspA’-pcDNA3.1 and PsaA-pcDNA3.1, alone or combined, than the contral plasmid and PBS groups. And the effect of immunization on carriage with mixed DNA vaccine plasmids was highly significant versus with one of them alone.
②The protection against S. pneumoniae D39 invasive infection. In the intraperitoneal-challenge experiments, we observed the corresponding result that mice immunizaed with the mixed DNA vaccines plasmids PspA’-pcDNA3.1+ PsaA-pcDNA3.1 had significantly longer median survival times than the group that received alone with one of them.
2. The immunogenicity and protection effects in mice immunized intramuscularly with oral S. pneumoniae multivalent DNA vaccines with the new carrier system.
⑴RT-PCR and Western- blot analysis of total cell extracts showed successful transcription and expression of pcDNA3.1001 and pcDNA3.1002 in BHK-21 cells. All of these plasmids(pcDNA3.1000, pcDNA3.1001, pcDNA3.1002) were 100% stably maintained for 25 generations in S. typhimurium (Asd-) hosts grown in the presence or absence of DAP.
⑵Immune responses in mice immunized with oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x.
①Cell immune responses. LPS antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with pcDNA3.1000x, pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x+pcDNA3.1002x were all significantly higher on 21th day after last immunization than that on day 0, but there had no significantly difference among all groups; PsaA or PspA antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with pcDNA3.1000x, pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x + pcDNA3.1002x were all significantly higher on 21th day after last immunization than that on day 0, and PsaA or PspA antigen-specific cytokine IFN-γin spleen cells supernatant of mice group immunized with oral DNA vaccines(pcDNA3.1001x, pcDNA3.1002x or pcDNA3.1001x+pcDNA3.1002x ) were significantly higher than DNA vaccine plamids(pcDNA3.101, pcDNA3.102 or pcDNA3.101+pcDNA3.102); PsaA and PspA’antigen-specific cytokine IL-4 in spleen cells supernatant of all mice group immunized were all low on day 0 or on 21th day after last immunization, and there had no significantly divergence between on day 0 with on 21th day after last immunization,or among all groups.
②Humoral immune responses. The antibody responses to Salmonella LPS and to the foreign antigen PspA’or PsaA in the sera and the nasal wash of the immunized mice were measured. Responses reached a maximal anti-LPS, -PsaA, and–PspA’IgG levels on 28th day after last immunization and all IgG antibody responses in vaccine groups were significantly stronger than the control pcDNA3.1 (+) and pcDNA3.1000x groups. Anti-LPS IgG antibody levels were similar in the mice of groups immunized by all oral DNA vaccines. IgG antibody levels against foreign antigens (PspA’or PsaA) in the mice of groups immunized by the oral multiantigen DNA vaccines ( pcDNA3.1001x+ pcDNA3.1002x)were significantly higher than that by the multiantigen DNA vaccine plasmids (i.m) (pcDNA3.101+ pcDNA3.102). IgG antibody levels against PspA’or PsaA in the mice of groups immunized by the multiantigen DNA vaccines (pcDNA3.1001x+pcDNA3.1002x or pcDNA3.101+ pcDNA3.102 )were significantly higher than that by the univalent DNA vaccines encoding either one of pspA’and psaA (including pcDNA3.1001x, pcDNA3.1002x and pcDNA3.101, pcDNA3.102).
③Mucosal immune responses. Secretory IgA antibodies to PspA’and PsaA were detected in the nasal washes of mice immunized by oral DNA vaccines, and negligible secretory IgA elicited by the DNA vaccine plasmids (i.m.) group. On 28th day after last immunization, the antibody leves reached peak, and higher IgA antibody levels against either PspA’or PsaA were observed in the groups of mice immunized with oral multiantigen DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) than in mice immunized with one of them alone (pcDNA3.1001x, pcDNA3.1002x) and the control group pcDNA3.1000x.
⑶Evaluation of levels of carriage and protective immunity in mice immunized intramuscularly with oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x.
①The protection against S. pneumoniae D39 nasopharyngeal colonization. The result showed fewer pneumococci were recovered from mice immunized with oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) than that immunited with the DNA vaccines (pcDNA3.101+ pcDNA3.102). The effect of immunization on carriage with mixed oral DNA vaccine pcDNA3.1001x +pcDNA3.1002x was highly significant versus with one of them alone pcDNA3.1001x or pcDNA3.1002x.
②The protection against S. pneumoniae D39 invasive infection. In the intraperitoneal-challenge experiments, we observed the corresponding result that mice immunizaed with oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) had significantly longer median survival times than that immunited with the DNA vaccines (pcDNA3.101+ pcDNA3.102). And the mice immunizaed with mixed oral DNA vaccines (pcDNA3.1001x+pcDNA3.1002x) had significantly longer median survival times than that immunited with one of them alone.
3. High frequency events of HCG and recombination support the prevalent presence of PsaA gene in streptococcus mitis group. These species may acquire transforming PsaA gene from the other species of streptococcus mitis group living in the same ecological niche, and maybe involved in promote the adhesion behavior and subsequent oropharyngeal colonization behavior.
4. PspA gene diversity of the region N terminal antigen genomic analysis suggested that the trend of regional distribution of PspA protein family: PspA-Fam1-Clade2 (31.0%) and PspA-Fam2-Clade3 (47.6%) as the predominant strains, and differences in foreign reports.
Conclusion
Successfully, oral S. pneumoniae multivalent DNA vaccines with the new carrier system pcDNA3.1001x and pcDNA3.1002x induced significant immune responses versus the DNA vaccine plasmids pcDNA3.101 and pcDNA3.102 intramuscular injected (i.m.), containing mucosa, cellular and humoral immune responses, and enhanced protection against nasopharyngeal colonization and protection against the intraperitoneal challenge. The studies also confirmed the cooperating, additive protection with multiantigen DNA vaccines against S. pneumoniae nasopharyngeal colonization and infection. The results provided the potential of using the salmonella-based balanced-lethal host-eukaryotic vector system as carriers to deliver S. pneumoniae protective antigens.
PsaA and PspA are the best forms of dominant antigen candidates of the new Streptococcus pneumonia generation vaccines. PsaA gene is vertical inheritance in Streptococcus pneumoniae, which may be aquired by other symbiotic streptococcus through horizontal gene transfer and recombinant gene manner. PspA gene has the diversity, and the target antigens should also include the clades components of Fam1 and Fam2 with high protective and strong cross-reaction against a wide range of clinical pathogenic strains of Streptococcus pneumoniae.
引文
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